Gi/o protein-coupled receptors inhibit neurons but activate astrocytes and stimulate gliotransmission

Abstract

G protein-coupled receptors (GPCRs) play key roles in intercellular signaling in the brain. Their effects on cellular function have been largely studied in neurons, but their functional consequences on astrocytes are less known. Using both endogenous and chemogenetic approaches with DREADDs, we have investigated the effects of G_q and G_i/o GPCR activation on astroglial Ca²⁺ -based activity, gliotransmitter release, and the functional consequences on neuronal electrical activity. We found that while G_q GPCR activation led to cellular activation in both neurons and astrocytes, G_i/o GPCR activation led to cellular inhibition in neurons and cellular activation in astrocytes. Astroglial activation by either G_q or G_i/o protein-mediated signaling stimulated gliotransmitter release, which increased neuronal excitability. Additionally, activation of G_q and G_i/o DREADDs in vivo increased astrocyte Ca²⁺ activity and modified neuronal network electrical activity. Present results reveal additional complexity of the signaling consequences of excitatory and inhibitory neurotransmitters in astroglia-neuron network operation and brain function.

Keywords: DREADDs; G protein-coupled receptors; astrocyte Ca2+; astrocytes; gliotransmission.

Figure 1.. G_q signaling induces neuronal activation.

(A) Scheme of neuronal G_q GPCR activation by ACh, and TexasRed and Fluo4-filled CA1 neuron (scale bar, 20µm). (B) Representative traces showing effects of local ACh application in current clamp (CC) and voltage clamp (VC). (C) ACh-induced current amplitude in different conditions (Kruskal-Wallis One-way ANOVA, p < 0.001). (D) Pseudocolor Fluo4 fluorescence images before and after ACh application in the neuron depicted in (A) (scale bar, 20µm), and corresponding fluorescent Ca²⁺ trace. (E) Percentage of neurons that responded with a Ca²⁺ increase to ACh in different conditions. (F) Immunohistochemical images of hippocampus injected with AAV5-CaMKIIα-hM3Dq-mCherry. From left to right: mCherry, NeuN, GFAP and merge (scale bar, 25 μm; s.o., stratum oriens; s.p., stratum pyramidale; s.r., stratum radiatum). (G) Scheme of neuronal chemogenetic G_qDREADD activation and image showing mCherry-expressing CA1 neurons (scale bar, 15 µm). (H) Representative traces showing effects of local CNO application in CC and VC. (I) CNO-induced current amplitude in different conditions (Kruskal-Wallis One-way ANOVA, p < 0.001). (J) Pseudocolor Fluo4 fluorescence image before and after CNO application (scale bar, 10µm), and corresponding fluorescent Ca²⁺ trace. (K) Percentage of neurons that responded with a Ca²⁺ increase to CNO in different conditions. Data are represented as mean ± SEM. P < 0.01 (**), and P < 0.001 (***).

Figure 2.. G_q activation in astrocytes elevates their Ca2+ levels.

(A) Scheme of G_q GPCR activation in astrocytes by ACh locally applied over stratum radiatum astrocytes. (B) Fluorescence images of an astrocyte showing selected domains in soma and processes (top), pseudocolor Ca²⁺ images before (basal) and after local application of ACh (bottom) (scale bar, 5 μm), and Ca²⁺ traces from domains shown in top right panel. (C) Left: Raster plot showing Ca²⁺ events for each ROI (somas (s) below reference line, processes (p) above reference line). Right: Average Ca²⁺ traces from responding somas (top) and processes (bottom). T-test compares 10s before and 10s after agonist. (D and E) Ca²⁺ event probability in somas (D) and processes (E) vs. time, and maximum values in different conditions. (F) Immunohistochemical images of AAV8-GFAP-hM3Dq-mCherry expression in the hippocampus. From left to right: Expression of mCherry, GFAP, NeuN, a merge of all three, and a higher magnification merge image showing colocalization of GFAP and mCherry (25x; scale bar, 20 μm; s.o., stratum oriens; s.p., stratum pyramidale; s.r., stratum radiatum). (G-K) as (A-E) but with local application of CNO to G_qDREADD expressing astrocytes instead of ACh. Data are represented as mean ± SEM. P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).

Figure 3.. G_i/o activation in neurons is inhibitory.

(A) Scheme of neuronal G_i/o activation by GABA, and TexasRed and Fluo4-filled CA1 neuron (scale bar, 20μm). (B) Representative traces showing GABA-induced responses in current clamp (CC) and voltage clamp (VC), and GABA-induced current amplitude in different conditions (Kruskal-Wallis One-way ANOVA, p < 0.01, p < 0.05). (C) Pseudocolor Fluo4 images before and after GABA application from the neuron depicted in (A) (scale bar, 20µm), and the corresponding fluorescent Ca²⁺ trace. (D) Immunohistochemical images of AAV5-CaMKIIα-hM4Di-mCherry expression in hippocampus. From left to right: mCherry, NeuN, GFAP and merge (scale bar, 50 μm; s.o., stratum oriens; s.p., stratum pyramidale; s.r., stratum radiatum). (E) Scheme of neuronal chemogenetic G_i/oDREADD activation and fluorescent image showing mCherry-expressing neurons (scale bar, 50µm). (F) Representative traces showing CNO-evoked responses in CC and VC, and CNO-induced current amplitude in different conditions (Kruskal-Wallis One-way ANOVA, p < 0.05). (G) Pseudocolor Fluo4 images before and after CNO application (scale bar, 20µm), and corresponding fluorescent Ca²⁺ trace. Data are represented as mean ± SEM. P < 0.05 (*) and P < 0.01 (**).

Figure 4.. G_i/o activation in astrocytes elevates their Ca2+ levels.

(A) Scheme of endogenous astrocyte G_i/o GPCR activation by GABA. (B) Fluorescence images of an astrocyte showing selected domains in soma and processes (top row), pseudocolor Ca²⁺ images before and after local application of GABA (bottom row; scale bar, 5 μm), and Ca²⁺ traces from domains shown in top left panel (right). (C) Left: Raster plot showing Ca²⁺ events for each ROI (somas (s) below reference line, processes (p) above reference line). Right: Average Ca²⁺ trace from responding somas (top) and processes (bottom). T-test compares 10s before and 10s after agonist. (D and E) Ca²⁺ event probability in somas (D) and processes (E) vs. time, and maximum values in different conditions. (F) Immunohistochemical images of AAV8-GFAP-hM4Di-mCherry expression in the hippocampus. From left to right: mCherry, GFAP, NeuN, a merge of all three, and a higher magnification merge image showing colocalization of GFAP and mCherry (25x; scale bar, 40 μm; s.o., stratum oriens; s.p., stratum pyramidale; s.r., stratum radiatum). (G-K) as (A-E) but with local application of CNO to G_i/oDREADD-expressing astrocytes instead of GABA. Data are represented as mean ± SEM. P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).

Figure 5.. G_i/o and G_q GPCRs stimulate different signaling pathways that do not occlude.

(A and B) Scheme of experimental set-up, and astrocyte Ca²⁺ responses to ACh (left), DHPG (middle), and both simultaneously (right), showing the observed (black trace) and expected (green trace) responses. Expected response corresponds to the linear summation of the responses evoked by independent application of ACh and DHPG. (C and D) as in (A and B), but with GABA instead of DHPG. (E) Histogram of the ratio of the observed and expected Ca²⁺ responses evoked by simultaneous application of GPCR agonists in the different pharmacological conditions. Expected responses were considered the linear summation of the responses elicited by independent stimulation of the agonists. Data are represented as mean ± SEM. P < 0.001 (***).

Figure 6.. Astrocyte activation via G_q and G_i/o DREADDs induces slow inward currents in neurons.

(A) Representative excitatory postsynaptic current (EPSC) and slow inward current (SIC) recorded from a CA1 pyramidal neuron. (B) Representative traces showing SICs (asterisks) in control and in D-AP5. (C) Scheme of neuronal-astroglial synaptic elements and gliotransmission. (D) Scheme and representative traces showing SICs (asterisks) before and after ACh application. (E) Left: Mean number of SICs vs. time, binned at 10s (ACh was applied at t=0). T-test compares one minute before and one minute after agonist. Right: SIC frequency (per min) one minute before and one minute after ACh in different conditions. Statistical significance was determined by the Student’s t-test comparing mean values one minute before and after agonist application. (F-G) as in (D-E) but with CNO application to G_qDREADD-expressing astrocytes. (H-I) as in (D-E) but with GABA application instead of ACh. (J-K) as in (D-E) but with CNO application to G_i/oDREADD-expressing astrocytes. Data are represented as mean ± SEM. P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).

Figure 7.. Astrocytic G_q and G_i/o DREADD activation increases neuronal action potential firing.

(A and B) Representative trace of spontaneous action potential firing of CA1 pyramidal neuron before and after CNO application to G_qDREADD-expressing astrocytes (A) and G_i/oDREADD-expressing astrocytes (B), and the normalized (from basal) firing frequency vs. time. CNO application was at t=0 (as in the other panels). (C and D) Representative trace (top) of action potential firing induced by short depolarizing pulses before and after CNO application to G_qDREADD-expressing astrocytes (C) and G_i/oDREADD-expressing astrocytes (D), expanded traces (bottom left panels), and normalized firing frequency vs. time (bottom right panels). (E and F) Response to CNO in the presence of D-AP5. (G and H) Normalized firing frequency before and after CNO application to G_qDREADD-expressing astrocytes (G) and G_qDREADD-expressing astrocytes (H) in control and D-AP5. Data are represented as mean ± SEM. P < 0.05 (*).

Figure 8.. In vivo activation of both G_q and G_i/o DREADDs in astrocytes elevates their Ca2+ levels and regulates neuronal electrical activity.

(A) Immunohistochemical representative image of G_qDREADDs virus injected into the primary somatosensory cortex staining for neurons (NeuN; blue), astroglia (GFAP; green), the reporter mCherry (red), and colocalization of mCherry with GFAP (top) and NeuN (bottom; scale bar, 20 μm). (B and C) Left: Representative images of mCherry in astrocytes reporting expression of G_qDREADDs and G_i/oDREADDs (scale bar, 50 μm). Pseudocolor Ca²⁺images before (middle) and after (right) intraperitoneal injection (i.p.) of CNO. (D and E) Raster plots of Ca²⁺ events before and after i.p. injection of CNO in mice with astrocytes expressing G_q and G_i/oDREADDs, respectively. (F and G) Representative Ca²⁺ traces during basal (left) and after i.p. CNO injection (right) in G_qDREADD- (F) and G_i/oDREADD-injected (G) mice. Ca²⁺ oscillation frequency normalized to baseline for DREADDs-infected cortex with i.p. CNO injections (solid bars), for DREADDs-infected cortex with i.p. saline injections (hashed bars), and for mCherry control-infected cortex with i.p. CNO injections (unfilled bars) for G_qDREADDs- (F) and G_i/oDREADDs-injected (G) mice. (H and I) Representative cortical local field potential recordings prior to CNO (left) and after (right) in G_qDREADDs- (H) and G_i/oDREADDs-injected (I) mice. Lowpass filtered at 40Hz. (J and K) Normalized power spectrum of cortical local field potentials before (black) and after CNO in G_qDREADDs- (J: blue) and G_i/oDREADDs-injected (K: red) mice. Normalized power spectrum of slow-wave delta (0–4 Hz) activity for DREADDs-infected cortex with i.p. CNO injections (solid bars), DREADDs-infected cortex with i.p. saline injections (hashed bars), and mCherry control-infected cortex with i.p. CNO injections for G_qDREADDs- (J) and G_i/oDREADDs-injected (K) mice. Error bars are SEM. P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).